J. Biochem., 82, 105-115 (1977)

A Chemically Modified Subfragment-l of from as a Novel Tool for Identifying the Function of Actomyosin in Non-Muscle Cells1

Masahiro ISHIURA,2 Kazuko SHIBATA-SEKIYA,2 Toyoki KATO, and Yuji TONOMURA

Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560

Received for publication, January 29, 1977

Two kinds of subfragment-1 of myosin, S-1(T) and S-1(CT), were prepared by two-step tryptic [EC 3.4.21.4] digestion of myosin that had been modified with about 1 mol of p-chloromercuri benzoate (CMB) per mol of myosin, and one-step chymotryptic [EC 3.4.21. 1] digestion of the myosin, respectively. The amount of bound CMB was about 0.82-0.90 mol per 2 mol of S-1. Both kinds of S-1 modified with CMB equally inhibited superprecipitation of myosin B from rabbit skeletal muscle. About 2 mol of CMB-S-1 (1 mol of CMB-S-IA) inhibited the function of 1 mol of monomer on the superprecipitation of actomyosin reconstituted from myosin and fibrous actin(FA) with relaxing (RP). CMB-S-1 also effectively inhibited superprecipitation of myosin B from the plasmodia of the slime mold Physarum polycephalum. The ATPase [EC 3.6.1.3] activity of CMB-S-1(T) was similar to that of CMB-S-1(CT) in the absence of FA, but was not enhanced as effectively by FA as the latter. In the presence of 0.3 mg/ml of FA with RP, the activity of CMB-S-1(T) was only one-fifth of that of CMB-S-1 (CT). CMB-S-1(T) did not affect the activities of ATPase from animal cells outside actomyosin systems, such as the Ca 2+-dependent ATPase [EC 3.6.1.3] of the SR prepared from rabbit skeletal muscle and the Na+,K+-dependent ATPase [EC 3.6.1.3] from porcine kidney. It also scarcely affected Ca 2+-uptake by the SR at concentrations lower than 0.2 mg/ml. However, CMB-S-1(T) strongly inhibited the polymerization and depolymerization of tubulin prepared from bovine brain. At 0.15 mol per mol of tubulin heterodimer, CMB-S-1(T)

1 This investigation was supported by a grant for the Japan-U.S. Cooperative Science Program from the Japan

Society for the Promotion of Science, and also by grants from the Ministry of Education, Science and Culture of Japan and the Muscular Dystrophy Association, Inc. 2 On leave from the Research Institute for Microbial Diseases, Osaka University, Yamada-Kami, Suita, Osaka 565.

3 Present address: Nayoro Women's College, Nayoro City, Hokkaido 096. Abbreviations: HMM, H-meromyosin; S-1, subfragment-1 of myosin; S-1(T), S-1 prepared by tryptic digestion

of HMM; S-1(CT), S-1 prepared by chymotryptic digestion of myosin; S-1B, S-1 with the initial burst of Pi-libera tion; S-lA, S-i without the Pi-burst; FA, F-actin; RP, relaxing protein; SR, sarcoplasmic reticulum; CMB, p

- chloromercuribenzoate; EGTA, ethyleneglycol his (ƒÀ-aminoethy]ether)-N,N•Œ-tetraacetate; Mes, 2-(N-morpholino) ethanesulfonic acid.

Vol. 82, No. 1, 1977 105 106 M. ISHIURA, K. SHIBATA-SEKIYA , T. KATO, and Y. TONOMURA

inhibited by 50% the extent of polymerization of 0.80 mg/ml tubulin (7.3 ƒÊm tubulin hetero

dimer). S-l(T) also inhibited tubulin polymerization as effectively as CMB-S-1(T). CMB

S-1(CMB-S-1A) also weakly bound itself to polymerized tubulin.

It was concluded that CMB-S-1(T) can be used as a specific inhibitor of actin functions in non-muscle cells if the possible involvement of tubulin is excluded by other means.

Actin, myosin, and tropomyosin have been isolated In the present paper, we describe experiments from many kinds of non-muscle cells and their showing that subfragment-l modified with CMB localization in cells has been studied (see review by (CMB-S-1) meets the requirement for an actin Pollard and Weihing, Ref. 1). Actin has been specific inhibitor. Previously, we showed that visualized in cells by selective decoration of actin CMB-S-1 is composed of equimolar S-lA modified filaments with HMM (2) or by specifically staining with CMB(CMB-S-lA) and S-1B not modified with them with fluorescent dye-labeled HMM (3). Re CMB, and that CMB-S-1 inhibits the superpre cently, antibodies specifically reactive with each cipitation of actomyosin induced by ATP, since component of contractile from non-muscle CMB-S-1A binds very tightly with FA even in the cells, such as actin (4), myosin (5), tropomyosin (6), presence of ATP, and thus occupies all the myosin and ƒ¿-actinin (7), have been prepared and used binding sites of actin (26). In the present work, successfully to show the intracellular distribution we prepared two kinds of CMB-S-1, CMB-S-1(T), of each component. Much of the actin is localized and CMB-S-l(CT), and compared their inhibition on microfilaments (2, 4), and in some cells, myosin of the superprecipitation of actomyosin and their

(5), tropomyosin (6), and ƒ¿-actinin (7) were also ATPase activities in the presence of FA. We associated with actin filaments. While no function found that the two kinds of CMB-S-1 inhibited has been established for these proteins, they have superprecipitation of actomyosin from rabbit been implicated in a variety of cell functions includ skeletal muscle with the same effectiveness, while ing motility, exocytosis, cytokinesis, membrane the acto-S-1-ATPase activity of CMB-S-i(T) was ruffling, maintenance of cell shape and cell adhesion much lower than that of CMB-S-l(CT). Thus, to substratum (8-13, see also Ref. 1 for review). using CMB-S-1(T), we studied the stoichiometric Cytochalasin B and D, microfilament-disrupt relation between CMB-S-1 and actin in the inhibi

ing mold metabolites, have been widely used to tion of superprecipitation by CMB-S-1, and found identify the functions of microfilaments in non that 2 mol of CMB-S-1 (1 mol of CMB-S-lA) muscle cells (13-19). However, it remains difficult inhibited the function of 1 mol of actin monomer.

to identify actin-specific functions in non-muscle To see the effectiveness of CMB-S-1 on actomyosin

cells, since there is no direct evidence showing that of non-muscle origin, we prepared Physarum myosin

cytochalasins specifically inhibit actin functions. B, and found that CMB-S-1 also effectively inhibited

Cytochalasin D was reported to bind with myosin the superprecipitation of Physarum myosin B. To

and inhibit myosin ATPase [EC 3. 6. 1. 3] (20). confirm the specificity of CMB-S-1, we prepared

Cytochalasin B has the undesirable side effect of SR [EC 3.6.1.3] from rabbit skeletal muscle, Na+, inhibiting some cellular transport systems (21-24). K+-dependent ATPase [EC 3.6.1.3] from porcine

To identify actin functions in non-muscle cells, kidney and tubulin from bovine brain, and studied

first, we must find a substance which binds with their possible interactions with CMB-S-1. We

actin specifically and stoichiometrically and inhibits found that they scarcely interacted with CMB-S-1

actin functions. Second, we must elaborate a except for tubulin. Therefore, we concluded that method of introducing the substance into cells CMB-S-1 can be used as a specific inhibitor of actin

without damaging cell functions. One such method although it may also interact with tubulin.

enables us to introduce any substance into a target cell by HVJ (Sendai virus)-mediated cell fusion EXPERIMENTAL between the target cell and a human erythrocyte ghost loaded with(25) the substance Materials-Myosin (MW=4.8 x 106) was pre

J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 107

pared from rabbit skeletal white muscle by the NaOH (pH 6.5), at 4•Ž. Next, it was homo

method of Perry (27). HMM (MW =3.4 x 105) genized in I ml of buffer per g of tissue with a was prepared by tryptic digestion of myosin by the Sorvall Omnimixer for I min at half-maximum

method of Szent-Gyorgyi (28) with slight modifi speed. The homogenate was centrifuged at

cation (29). S-i(T) (MW=1.2 x 105) was prepared 100,000 x q for 1 h at 4•Ž. The supernatant was

by tryptic digestion of HMM, followed by chro collected, and mixed with an equal volume of the

matography on Sephadex G-200, as described reassembly buffer containing 8 M glycerol, and

previously (26, 30). S-1(CT) (MW=1.2 x 105) was incubated for 20 min at 37•Ž to polymerize tubulin. prepared by chymotryptic digestion of myosin at Polymerized tubulin was collected by 100,000 x g low ionic strength, as described by Weeds and centrifugation for I h at 25•Ž, then dissolved in

Taylor (31), and purified on Sephadex G-200. cold reassembly buffer, which contained 1 mm GTP G-actin with RP was extracted from an acetone instead of ATP (GTP-reassembly buffer), by gentle

powder of rabbit skeletal muscle at room tem homogenization in a glass homogenizer. Next, perature, and purified by a polymerization-depoly the mixture was chilled on ice for 30 min to de

merization procedure (32). Purified G-actin was polymerize tubulin. The tubulin solution was

prepared from an acetone powder of rabbit skeletal clarified by 100,000 x g centrifugation for 1 h at muscle by the method of Spudich and Watt (33). 4•Ž. The polymerization-depolymerization cycle

After removal of free nucleotides from the G-actin was repeated twice in the presence of 1 mm GTP.

solution by treatment with Dowex 1 •~4, actin was After the second polymerization, the dissociated

polymerized by addition of 1 mm MgCl: and 50 mm tubulin in GTP-reassembly buffer was brought to KCl. 8 M in glycerol and stored at -20•Ž, unless other

Myosin B was prepared from rabbit skeletal wise stated. The tubulin was more than 70% pure

muscle by the method described by Szent-Gyorgyi according to electrophoresis on sodium dodecyl

(34) with slight modification. Cat+-sensitive sulfate-polyacrylamide gels performed as described Physarum myosin B was prepared from the plasmo previously (30); it was contaminated mainly with dia of the slime mold, Physarum polycephalum, as tubulin-associated high molecular weight compo

described previously (35). nents.

SR was prepared from rabbit skeletal muscle, Pyruvate kinase [EC 2.7.1.40] was prepared

as described previously (36). The activity of Ca 2+_ from rabbit skeletal muscle by the method of Tietz dependent ATPase of the SR was responsible for and Ochoa (39).

more than 97 % of the total ATPase activity of the Trypsin was purchased from Worthington

SR. The preparation used was kindly supplied by Chemical Co. Trypsin inhibitor and chymotryp

Dr. T. Yamamoto sin were purchased from Sigma Chemical Co.

. Na+, K+-dependent ATPase was prepared from Bovine serum albumin (fraction 5) was purchased

the dark outer medulla of porcine kidney by the from Nakarai Chemicals, Ltd. 203Hg-CMB and

method of Jorgensen (37) with slight modification. CMB were purchased from the Radiochemical

The ATPase activity of the enzyme preparation Centre, England, and Wako Pure Chemical

was usually more than 2,000 umol per mg protein Industries, Ltd., respectively, and purified by Bo yer's method (40). 4bCaCl2 was purchased from per h at 37•Ž, and completely inhibited by ouabain the Radiochemical Centre. Phenylmethylsulfonyl (M. Yamaguchi & Y. Tonomura, unpublished fluoride and phosphoenolpyruvate were purchased observation). The enzyme was a generous gift from Sigma Chemical Co., and ƒÀ-mercaptoethanol from Mr. M. Yamaguchi. from Nakarai Chemicals, Ltd. GTP and ATP Bovine brain tubulin (ƒ¿ƒÀ-heterodimer, MW= were purchased from P.L. Biochemicals Inc. and 1.1 x 105) was purified from fresh brains essentially Kyowa Hakko Co., respectively. by the assembly-disassembly procedure described Preparation of CMB-S-1 (T) and CMB by Shelanski et al. (38). The superficial blood S-1(CT)-Myosin was modified with CMB as vessels were removed, then the brain was washed described previously (26). CMB-HMM was pre with 0.34 M sucrose and minced with scissors in pared by tryptic digestion of CMB-myosin. CMB ATP-reassembly buffer, which contained 1 mm S-l(T) and CMB-S-1(CT) were prepared by tryptic ATP, 1 mm EGTA, 0.5 mm MgCl2, and 0.1 M Mes

Vol. 82, No. 1, 1977 108 M. ISHIURA, K. SHIBATA-SEKIYA, T. KATO, and Y. TONOMURA digestion of CMB-HMM and chymotryptic diges muscle. tion of CMB-myosin, respectively. The radio When superprecipitation was initiated in the activity of 203Hg-CMB bound to S-1 was measured presence of CMB-S-1(T) or CMB-S-1(CT), both in a Beckman liquid scintillation spectrometer, inhibited the rate and extent of superprecipitation Model SL 150. CMB-S-1 contained 0.82-0.9 mol of myosin B. Figure 1 shows that the two kinds of CMB per 2 mol of S-1. of CMB-S-1 inhibited the extent of superprecipita

ATPase Activity and Ca2+-Uptake-The ATPase tion with same efficiency. This suggests that the activity of acto-S-1 in the steady state was de tryptic and chymotryptic digestion for the prepara termined at 25°C in a coupled system, with pyruvate tion of CMB-S-1(T) and CMB-S-1(CT), respec kinase and phosphoenolpyruvate as an ATP tively, did not damage the inhibitory function of regenerating system, as described previously (29). myosin head modified with CMB. S-1 not modi

The Ca 2+-dependent ATPase activity of SR was fied with CMB, such as S-1(T) or S-1(CT), did not determined at 25°C as described previously (41). affect the superprecipitation of 0.28 mg/ml myosin The amount of Ca2+-uptake by SR was measured B at the concentration of 0.6 mg/ml. by the Millipore filtration method using 45CaCl2 as The ATPase activities of the two kinds of substrate, as described previously (41, 42). The CMB-S-1 were measured at 25°C in the presence activity of Na+, K+-dependent ATPase was of 0-0.6 mg/ml FA with RP and 50 mm KCl at measured as described by Jorgensen (37). pH 7.6. Their ATPase activities in the absence of Superprecipitation of Actomyosin and Myosin FA were about 50-100 umol per min per g of S-1. B-The superprecipitation of actomyosin and The activity of CMB-S-1(CT) gradually increased myosin B was measured at 20°C by determining with increasing concentration of FA with RP, and turbidity at 660 nm with a Cary Model 14 spectro the activities in the presence of 0.3 and 0.6 mg/ml photometer, as described previously (26). FA with RP were 780 and 1,060 ƒÊmol per min per Polymerization of Tubulin-The polymeriza g of S-l, respectively. However, FA did not tion of tubulin in GTP-reassembly buffer was enhance the activity of CMB-S-1(T) as effectively measured at 25°C by the turbidimetric method described by Gaskin et al. (43). The time course of increase in absorbance at 350 nm was measured with a Zeiss Model PMQ 3 spectrophotometer. Protein Concentration-Protein concentration was estimated by the biuret reaction (44) calibrated by nitrogen determination, or by the Copper-Folin method (45).

RESULTS AND DISCUSSION

Stoichiometric Inhibition of the Superprecipita tion of Actomyosin by CMB-S-1-S-1(CT) holds the myosin-associated functions of interacting with actin, such as the activation of S-1-ATPase activity Fig. 1. Inhibition of the superprecipitation of rabbit by FA and the acceleration of actin polymerization skeletal muscle myosin B by CMB-S-l(T) and CMB by S-1, more persistently than S-1(T) (46). We S-l(CT). Rabbit skeletal muscle myosin B (0.28 mg/ml) was mixed with various amounts of CMB-S-1(T) or prepared two kinds of S-1 modified with CMB, CMB-S-1(CT), and preincubated at 20°C for 5 min. CMB-S-l(T), and CMB-S-1(CT), and compared The clearing of myosin B was induced by adding 0.2 mm their inhibition of the superprecipitation of acto ATP in the presence of 62.5 mm KCl, 2 mm MgCl2, myosin. Since myosin B contains all the protein 1 mm EGTA, and 20 mm Tris-maleate (pH 7.0), and components of the muscle contractile system and is 1 min later, superprecipitation of myosin B was initiated considered to be a more native contractile model by adding 3 mm CaCl2. JA was defined as the final than actomyosin reconstituted from myosin and difference of absorbance at 660 nm in the presence and FA, we used myosin B prepared from rabbit skeletal absence of CaCl2. •ü, CMB-S-1(T); •œ CMB-S-1(CT).

J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 109

as that of CMB-S-l(CT). The activities of CMB S-1(T) in the presence of 0.3 and 0.6 mg/ml FA with RP were only 160 and 240 femol per min per g of S-1, respectively. This fact shows that the tryptic digestion of CMB-myosin somehow damaged myosin molecules, decreasing the efficien cy of FA activation of ATPase activity of CMB S-1(T). When CMB-S-1 is used as a specific inhibitor of actomyosin motile systems, CMB-S-1 - associated enzyme activities, such as ATPase, should be very low. We preferred CMB-S-1(T) as a candidate for a specific inhibitor of actomyosin motile systems. CMB-S-1 inhibited the superprecipitation of actomyosin reconstituted from pure FA and myosin as well as that of actomyosin reconstituted from FA with RP and myosin. In addition, this inhibited superprecipitation was recovered by addition of pure FA, but not by addition of other protein com ponents of myosin B. These facts clearly show that CMB-S-1 acted on actin to inhibit super precipitation of myosin B. Previously, we con Fig. 2. Stoichiometric inhibition of the superprecipita cluded that CMB-S-1(T) contains equimolar S-lA tion of rabbit skeletal muscle actomyosin by CMB modified with CMB(CMB-S-1 A) and unmodified S-1(T). Actomyosin was reconstituted from 0.6 mg/ml S-tB, and that only CMB-S-1 binds with FA tightly rabbit skeletal muscle myosin and various amounts of rabbit skeletal muscle FA with RP, and then mixed with in the presence of ATP (26). To show the stoichio various amounts of CMB-S-I(T). Other conditions for metric inhibition of actin function by CMB-S-1 on the superprecipitation of actomyosin were the same as the superprecipitation of actomyosin, we re in Fig. 1. •ü, Amount of CMB-S-1(T) required for half constituted actomyosin from 0.6 mg/ml of myosin inhibition of the extent of superprecipitation; •œ that and various amounts of FA with RP, and studied required for complete inhibition. the quantitative relation between the actin content in the reconstituted actomyosin and the amount of CMB-S-1(T) required to inhibit superprecipita Inhibition of Superprecipitation of Myosin B tion. Prepared from Non-Muscle Cells by CMB-S-1 Figure 2 shows the stoichiometric inhibition CMB-S-1(T) inhibited superprecipitation of acto of superprecipitation of actomyosin by CMB myosin prepared from non-muscle cells as well as S-1(T). The amounts of CMB-S-I(T) required for that of skeletal muscle actomyosin. Figure 3 shows half and complete inhibition of the extent of that CMB-S-1(T) inhibited both the rate and extent superprecipitation increased linearly with the of superprecipitation of Physarum myosin B. At amount of FA with RP in actomyosin. Assuming the concentrations of 0.24 and 0.74 mg/ml, CMB that the molecular weight of FA with RP per actin S-1(T) caused half and complete inhibition of the monomer is 6.3 x 10°, the amounts of CMB-S-1(T) extent of superprecipitation of 1 mg/ml Physarum required for the half and complete inhibition were myosin B, respectively. about 1.1 and 1.8 mot per mot of actin monomer, Effects of CMB-S-1 on the Activities of Ca2+ respectively. This fact clearly shows that 2 mot of - Dependent A TPase of SR and Na+, K+-Dependent CMB-S-1(T) (1 mot of CMB-S-lA) inhibited the ATPase Prepared from Porcine Kidney-To under function of 1 mot of actin monomer. Therefore, stand the specific inhibition of actin functions by we can conclude that I mot of CMB-S-IA binds to CMB-S-1, we studied the possible interaction of 1 mot of actin monomer and inhibits the functional CMB-S-1(T) with other ATPase prepared from interaction between actin and myosin. animal cells. Figure 4 shows that the Cat+-ATPase

Vol. 82, No. 1, 1977 110 M. ISHIURA, K. SHIBATA-SEKIYA, T. KATO, and Y. TONOMURA

Fig. 3. Inhibition of the superprecipitation of Physarum myosin B by CMB S-1(T). Physarum myosin B (1 mg/ml) was mixed with various amounts of CMB-S-1(T) and preincubated for 5 min at 20°C. Next, the clearing and super precipitation of Physarum myosin B were initiated by adding 0.2 mm ATP and 3 mm CaCl2, respectively, in the presence of 75 mm KCl, 2 mm MgCl2, 1 mm EGTA, and 20 mm imidazole-HCl (pH 7.0), as described in Fig. 1. Other conditions were the same as in Fig. 1. JA was defined as the increase of absorbance at 660nm. A. Time course of superprecipitation. CMB-S-1(T) added: 0, 0 mg/ml; x, 0.5 mg/ml. I, addition of 1 mm ATP; y , addition of 3 mm CaCl2. B. Extent of superprecipitation. activity of SR (50 ƒÊg/m1 of SR protein) measured When the Ca 2+-uptake by SR was measured in the presence of 790 fig/ml CMB-S-1(T) was 93 in the presence of various amounts of CMB-S-1(T), of that in the absence of CMB-S-1(T). This fact it was scarcely affected at concentrations lower than suggests that there was no interaction of CMB 0.2 mg/ml, but partially inhibited with increasing

S-1(T) with the Ca2t-ATPase of SR. This view concentration (Fig. 5). Ca t+-uptake in the pres was supported by the following result of Cat+ ence of 0.53 and 0.79 mg/ml CMB-S-1(T) was 74 uptake experiment. and 47% of that in the absence of CMB-S-1(T),

J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 111

Fig. 5. Effect of CMB-S-1(T) on the Ca 2+-uptake Fig. 4. Effect of CMB-S-1(T) on the Ca2+-dependent activity of SR. Ca 2+-uptake by SR (50 ƒÊg/ml protein) ATPase activity of SR. ATPase activities of SR and/or was started by adding 2 mm ATP, and measured by CMB-S-1(T) were measured in the presence of 2 mm the Millipore filtration method in the presence of ATP, 70 mm KCl, 100 teM CaCl2, 5 mm MgCl2, and 2 mm ATP, 70 mm KCl, 100 ƒÊM 45CaCl2, 5 mm MgCl2, 60 mm Tris-maleate at pH 6.5 and 25•Ž. •ü, SR (50 and 60 mm Tris-maleate at pH 6.5 and 25•Ž. ƒÊ g/ml SR protein); x, CMB-S-1(T) (790 ƒÊg/ml); •œ SR (50 ƒÊg/ml SR protein) and CMB-S-1(T) (790 ƒÊg/ml); •ü +x.

respectively. The inhibition observed at high

CMB-S-1(T) concentrations would be caused

partially by the disturbing effect of high protein concentration on the Millipore filtration method,

since protein concentrations higher than 0.5 mg/ml

decrease the measured values of Call-uptake by

SR. CMB-S-l(T) also did not alter the activity of

Na+, K+-dependent ATPase prepared from porcine

kidney. Figure 6 shows that the Na+, K+-de

pendent ATPase activity measured in the presence of 100 ƒÊg/ml CMB-S-1(T) was 94% of that in the absence of CMB-S-1(T).

Interaction of CMB-S-1(T) with Tubulin-Tbe Fig. 6. Effect of CMB-S-1(T) on the activity of tubulin-dynein system commonly exists in animal Na+,K+-dependent ATPase from porcine kidney. cells, and is believed to be involved in various cell ATPase activities of Na+,K+-dependent ATPase and/or associated phenomena, such as cell division, cell CMB-S-1(T) were measured in the presence of 2 mm motility, transport, pinocytosis and secretion. In ATP, 140 mm NaCl, 15 mm KCl, 5 mm MgCl2, and

addition, some authors recently reported that 206 mM Tris-HCl at pH 7.6 and 37•Ž. •ü, Na+,K+

colchicine-binding protein from porcine brain (47) dependent ATPase (680 ng/ml); x, CMB-S-1(T) (100 and blood platelets (48), and tubulin isolated from ƒÊg/ml); •œ Na+,K+-dependent ATPase (680 ng/ml) and CMB-S-1(T) (100 ƒÊg/ml); ----, •ü + X. the outer fiber of the flagella of Tetrahymena (49)

Vol. 82. No. 1, 1977 112 M. ISHIURA, K. SHIBATA-SEKIYA, T. KATO, and Y. TONOMURA

activate the Mg2+-ATPase activity of skeletal preparation since our tubulin was at least 70 % pure muscle myosin as actin does, and suggested a and showed no actin band on electrophoresis on

homology between tubulin and actin. Thus, we sodium dodecyl sulfate-polyacrylamide gels.

examined the possible interaciton of CMB-S-1(T) CMB-S-1 not only bound to polymerized with tubulin. tubulin but also inhibited polymerization of tubulin

Figure 7 shows the binding of CMB-S-1(T) to (Fig. 8). Both CMB-S-1(T) and S-1(T) strongly polymerized tubulin. When various amounts of inhibited polymerization of tubulin with the same 203Hg-CMB-S-1(T) were centrifuged with 1 .05 mg/ efficiency. They affected the polymerization of

ml of tubulin (9.5 ƒÊM tubulin heterodimer), a mix 0.80 mg/ml tubulin (7.3 ƒÊM tubulin heterodimer) at

ture of polymerized (74% of total tubulin) and concentrations greater than 45 ƒÊg/ml, and 0.12 unpolymerized tubulin, the radioactivity due to mg/ml of CMB-S-l(T) or S-1(T) caused 50% inhibi 203Hg-CMB bound to CMB-S-lA that coprecipita tion. The amount of CMB-S-1(T) or S-I(T) re

ted with polymerized tubulin by ultracentrifugation quired for half inhibition was 0.15 mol per mol of became greater with increasing 203Hg-CMB-S-1A tubulin heterodimer. The extent of the inhibition

concentration, although it was low even in the by CMB-S-1(T) or S-1(T) did not exceed about

presence of excess CMB-S-1A. Only 0.22 mol of 74%, which was observed at the concentration of CMB-S-1A per mol of tubulin heterodimer co 1 mg/ml. Bovine serum albumin did not affect the

precipitated with polymerized tubulin in the pres polymerization at 1 mg/ml. ence of 20ƒÊm CMB-S-1A. The coprecipitation of CMB-S-1(T) also inhibited depolymerization of CMB-S-1A with polymerized tubulin could not be polymerized tubulin initiated by addition of 2 rum attributed to actin contamination in the tubulin CaCl2 in GTP-reassembly buffer at 25•Ž. These

observations indicate that myosin head (at least head A) could interact with tubulin in vitro, and

suggest that the modification of head A with CMB

did not alter the interaction between myosin head and tubulin.

Fig. 7. Coprecipitation of CMB-S-1(T) with poly merized tubulin by ultracentrifugation. Tubulin (1.05 mg/m1=9.5 ƒÊM tubulin heterodimer) was incubated for 20 min at 37•Ž in GTP-reassembly buffer (1 mm GTP 0.5 mM MgCl2-0.1 M Mes-NaOH at pH 6.5) for poly merization of tubulin. Under these conditions, 74%

(7.0 ƒÊM) of total tubulin polymerized. The tubulin was mixed with various amounts of 203Hg-CMB-S-1(T) in Fig. 8. Effect of CMB-S-1(T) on tubulin polymeriza GTP-reassembly buffer and centrifuged at 100,000 x g tion. Tubulin (0.80 mg/m1=7.3 ƒÊM tubulin hetero for 1 h at 25•Ž. 203Hg-CMB-S-1(T) and the tubulin dimer) was mixed with various amounts of CMB-S-1(T) were also centrifuged separately. The amounts of or S-1(T), and preincubated for 5 min at 4•Ž in GTP 203Hg-CMB in the supernatant and precipitate were reassembly buffer. Polymerization was initiated by in measured by the radioactivity. The amount of 203Hg cubation at 25•Ž, and measured at 25•Ž in GTP

CMB coprecipitated with polymerized tubulin was reassembly buffer by the turbidimetric method. JA was calculated by subtracting the amount of 20°Hg-CMB defined as the increase of absorbance at 350 nm during the incubation for 60 min at 25•Ž. •ü, CMB-S-1(T); precipitated in the absence of tubulin from that in its presence. •œ S-1(T).

J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 113

The mechanism for inhibition of tubulin poly ing of the actin filaments using actin-specific anti merization and depolymerization by CMB-S-1(T) body (4), and actin is tentatively located on micro and S-1(T) is not clear. Tubulin requires equi filaments. Actomyosin in non-muscle cells is molar GTP for polymerization (50). The GTPase believed to be involved in various cell functions (1), activity associated with CMB-S-1(T) or S-1(T) mainly because the functions are inhibited by cyto could not account for the inhibition of tubulin chalasin B or D, which disrupts the organization of

polymerization, since the activity was not high microfilaments (13-19). However, actin functions enough to induce GTP depletion, which would in non-muscle cells are not yet clear mainly due to

inhibit the polymerization. When CMB-S-1(T) or the lack of an actin-specific inhibitor.

S-I(T) was incubated in GTP-reassembly buffer at Pure S-1, modified with CMB, is easily pre 25•Ž (conditions of the polymerization experiment), pared in large amounts from rabbit skeletal muscle.

0.12 mg/m1 of CMB-S-1(T) and S-1(T), which CMB-S-lA binds with FA stoichiometrically at a

inhibited by 50 % the polymerization of 0.80 mg/ml ratio of 1 mol of CMB-S-IA per mol of actin mono tubulin (Fig. 8), consumed only 25 and 39%. of the mer, and very tightly even in the presence of ATP.

total GTP (1 mm) per 30 min, respectively, while Thus it occupies all the myosin binding sites of

the polymerization was almost complete within actin, and inhibits the actin function to interact with

30 min under the given conditions. Polymeriza myosin. Therefore, we can estimate the content tion was inhibited 50 % by 0.15 mol of CMB-S-1 (T) of functional actin in cells using the specific binding

or S-I(T) per mol of tubulin heterodimer (Fig. 8). of 11314g-CMB-S-1 with actin in the presence of

This amount of CMB-S-1(T) is equivalent to 0.07 ATP. The distribution of actin within a cell can mol of CMB-S-1A per mol of tubulin heterodimer, also be seen under a fluorescence microscope using

provided that 2 mol of CMB-S-1(T) contained CMB-S-1 conjugated with a fluorescent dye, or 0.9 mol of CMB. Thus, in the presence of 9.5 ƒÊM using CMB-S-1 together with fluorescent dye

tubulin, 0.7 ƒÊM of CMB-S-1A caused 50% inhibi conjugated antibody reactive with S-1. Although tion of tubulin polymerization. At the concen HMM conjugated with fluorescein isothiocyanate

trations of tubulin and CMB-S-1A, only 0.02 mol has been successfully used to show the distribution

of CMB-S-IA per mol of tubulin heterodimer of actin within a cell (3), CMB-S-1 may be preferable bound to polymerized tubulin (Fig. 7). This since the binding of S-lA to FA is greatly strength

calculation shows that if the binding of CMB-S-lA ened by the modification with CMB (26). We can

(or S-lA) to tubulin inhibited tubulin polymeriza also identify the actin functions in non-muscle cells tion, the binding of only 0.02 mol of CMB-S-IA using CMB-S-1 as a specific inhibitor of actin.

(or S-lA) per mol of tubulin heterodimer could The ATPase activity associated with CMB-S-1(T) inhibit the polymerization by 50%. If so, we may is low even in the presence of FA, and thus CMB consider two possible mechanisms for the inhibi S-1(T) might affect the intracellular ATP level only

tion. One is that CMB-S-IA (or S-IA) binds to slightly if introduced into a cell. If large amounts

polymerized tubulin and blocks the elongation and of CMB-S-1A are isolated from CMB-S-1, it is shortening of the tubulin polymer. The other is preferable to CMB-S-1(T), since the ATPase ac

that CMB-S-1A (or S-1A) binds with microtubule tivity of CMB-S-1 A is not accelerated by FA (26). associated components that control the tubulin There is little possibility that CMB bound to S-1 is

polymerization and depolymerization (51-54), and transferred to intracellular components and affects affects the control functions of the components. the functions of components other than actin, since Further investigations are necessary to clarify the CMB bound to S-1 is difficult to remove from CMB

interaction between S-I and tubulin. S-1 even by treatment with ditiothreitol, and as reported previously (26), there is no indication of intramolecular and intermolecular transfer of CMB CONCLUSION bound to S-1 during the preparation of CMB-S-1 from CMB-myosin and during incubation of CMB Actomyosin exists commonly in various non-muscle S-1 with FA. CMB-S-I(T) does not interact with cells (1). The intracellular distribution of actin is easily seen by specific decoration of the actin fila a cell-associated ATPase, such as the Ca2+-depend ments with HMM (2) or immunofluorescence stain ent ATPase of SR from rabbit skeletal muscle or

Vol. 82, No. 1, 1977 114 M. ISHIURA, K. SHIBATA-SEKIYA, T. KATO, and Y. TONOMURA

the Na+, K+-dependent ATPase from porcine line from human amnions, is about 4 h (Yamaizumi, kidney. Since both CMB-S-1(T) and S-1(T) show M., Uchida, T., Furusawa, M., & Okada, Y., in some interaction with tubulin from bovine brain, preparation). we can interpret the inhibition of cell functions by

CMB-S-1 as a result of specific inhibition of actin We thank Dr. T. Yamamoto and Mr. M. Yamaguchi

functions, only when S-1 or a tubulin-specific for kindly suppling the preparations of SR and Na+,K+

inhibitor such as colchicine does not affect the same dependent ATPase and their advice on measuring the functions, excluding the possible involvement of activities of the preparations. M.I. thanks Dr. K. Take

tubulin in the functions. Furthermore, if the effect uchi, Dr. A. Inoue, and Mr. H. Takenaka for their kind advice, and Prof. Y. Okada for his guidance and en of S-1 to a cell function is dependent on the modifi couragement. cation of S-1 with CMB, then the involvement of

actomyosin, but not tubulin, in the function is highly probable, since the interaction of S-1 with REFERENCES tubulin is independent of the modification. 1. Pollard, T.D. & Weihing, R.R. (1973) CRC Critical Kuroda has already used CMB-S-1 successfully Reviews in 2, 1-65 to show the involvement of the actomyosin motile 2. Ishikawa, H., Bischoff, R., & Holtzer, H. (1969) system in the protoplasmic streaming in Nitella cells J. Cell Biol. 43, 312-328

(Kuroda, K., unpublished observation). She made 3. Sanger, J.W. (1975) Proc. Natl. Acad. Sci. U.S. 72, Nitella drops permeable to proteins by treatment 1913-1916

with EGTA, and showed that only 4ƒÊM CMB-S-l 4. Lazarides, E. & Weber, K. (1974) Proc. Nat!. Acad.

completely inhibits the rotation of chloroplasts Sci. U.S. 71, 2268-2272 within a Nitella drop as 1 mm N-ethylmaleimide, 5. Weber, K. & Groeschel-Stewart, U. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4561-4564 an SH reagent, does. 6. Lazarides, E. (1975) J. Cell Biol. 65, 549-561 We can introduce CMB-S-1 into human 7. Lazarides, E. & Burridge, K. (1975) Cell 6, 289-298 erythrocytes (HRBC) by procedures described 8. Buckley, LK. & Porter, K.R. (1967) Protoplasma 64, previously (25). A small amount of actin exists in 349-380 HRBC, associated with spectrin (55), but actin 9. Goldman, R.D. & Knipe, D.M. (1972) Cold Spring functions in HRBC are not clear. Since there is Harbor Symp. Quant. Biol. 37, 523-534

no indication of the existence of tubulin in HRBC, 10. Orr, T.S.C., Hall, D.E., & Allison, A.C. (1972)

the HRBC ghost loaded with CMB-S-1 is a very Nature 236, 350-351

useful tool for studying actin functions in erythro 11. Schroeder, T.E. (1970) Z. Zellforsch. Mikrosk. Anat. 109,431-449 cytes. Investigations on this line are in progress. 12. Spooner, B.S., Yamada, K.M., & Wessells, N.K. CMB-S-1 also can be introduced into animal cells (1971) J. Cell Biol. 49, 595-613 by HVJ (Sendai virus)-mediated cell fusion between 13. Wessells, N.K., Spooner, B.S., Ash, J.F., Bradley, a target cell and an HRBC ghost loaded with CMB M.O., Luduena, M.A., Taylor, E.L., Wrenn, J.T., S-1 (25). Successful inhibition of actin functions & Yamada, K.M. (1971) Science 171, 135-143 in non-muscle cells by introducing CMB-S-1 into 14. Auerberg, N. (1972) J. Cell Biol. 52, 206-211 the cells would depend on the efficiency of introduc 15. Carter, S.B. (1972) Endeavour 31, 77-81 tion into the target cell and the actin content in the 16. Wrenn, J.T. & Wessells, N.K. (1970) Proc. Natl.

cell, since the actin content in non-muscle cells of Acad. Sci. U.S. 66, 904-909

animal origin is rather high (0.2-10% of the 17. McGuire, J. & Moellmann, G. (1972) Science 175, 642-644 total cell proteins) (56-58). In addition, we must 18. Bradley, M.O. (1973) J. Cell Sci. 12, 327-343 take into account the intracellular proteolysis of 19. Spudich, J.A. (1973) Cold Spring Harbor Symp. CMB-S-1 introduced into a target cell, since a Quant. Biol. 37, 585-593 foreign protein introduced into a target cell is 20. Puszkin, E., Puszkin, S., Lo, L.W., Tanenbaum, degraded rather rapidly-the half life of the activity S.W. (1973) J. Biol. Chem. 248, 7754-7761 of bacteriophage T4-endonuclease V introduced 21. Estensen, R.D. & Plagemann, P.G.W. (1972) Proc. into the fibroblasts derived from Xeroderma pig Natl. Acad. Sci. U.S. 69,1430-1434

mentosum patients is about 3 h (59), and that of 22. Mizel, S.B. & Wilson, L. (1972) J. Biol. Chem. 247,

bovine serum albumin in FL cells, an established 4102-4105

J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 115

23. Kletzien, R.F., Perdue, J.F., & Springer, A. (1972) 41. Yamada, S., Yamamoto, T., & Tonomura, Y. (1970) J. Biol. Chem. 247, 2964-2966 J. Biochem. 67, 789-794 24. Kletzien, R.F. & Perdue, J.F. (1973) J. Biol. Chem. 42. Sumida, M. & Tonomura, Y. (1974) J. Biochem. 75, 248,711-719 283-297 25. Furusawa, M., Yamaizumi, M., Nishimura, T., 43. Gaskin, F., Cantor, C.R., & Shelanski, M.L. (1974) Uchida, T., & Okada, Y. (1976) in Methods in Cell J. Mol. Biol. 89,737-758 Biology (Prescott, D.M., ed.) Vol. 14, pp. 73-80, 44. Gornall, A.G., Bardawill, C.J., & David, M.M. Academic Press, Inc., New York (1949) J. Biol. Chem. 177, 751-766 26. Shibata-Sekiya, K. & Tonomura, Y. (1976) J. Bio 45. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & chem. 80, 1371-1380 Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 27. Perry, S.V. (1955) in Methods in Enzymology (Colo 46. Onodera, M. & Yagi, K. (1971) J. Biochem. 69, wick, S.P. & Kaplan, N.D., eds.) Vol. 2, pp. 582-588, 145-153 Academic Press, Inc., New York 47. Puszkin, S. & Bert, S. (1970) Nature 225, 558-559 28. Szent-Gyorgyi, A.G. (1953) Arch. Biochem. Biophys. 48. Puszkin, E., Puszkin, S., & Aledort, L.M. (1971) 42,305-320 J. Biol. Chem. 246, 271-276 29. Sekiya, K., Takeuchi, K., & Tonomura, Y. (1967) 49. Alicea, H.A. & Renaud, F.L. (1975) Nature 257, J. Biochem. 61, 567-579 601-602 30. Hayashi, Y. (1972) J. Biochem. 72, 83-100 50. Olmsted, J.B. & Borisy, G.G. (1975) Biochemistry 31. Weeds, A.G. & Taylor, R.S. (1975) Nature 257, 14,2996-3005 54-56 51. Weingarten, M.D., Lockwood, A.H., Hwo, S., & 32. Mommaerts, W.F.H.M. (1952) J. Biol. Chem. 198, Kirschner, M.W. (1975) Proc. Natl. Acad. Sci. U.S. 445-457 72,1858-1862 33. Spudich, J.A. & Watt, S. (1971) J. Biol. Chem. 246, 52. Murphy, D.B. & Borisy, G.G. (1975) Proc. Natl. 4866-4871 Acad. Sci. U.S. 72, 2696-2700 34. Szent-Gyorgyi, A. (1951) A Chemistry of Muscle 53. Keates, R.A.B. & Hall, R.H. (1975) Nature 257, Contraction 2nd ed., Academic Press, New York 418-420 35. Kato, T. & Tonomura, Y. (1975) J. Biochem. 77, 54. Borisy, G.G., Marcum, J.M., Olmsted, J.B., 1127-1134 Murphy, D.B., & Johnson, K.A. (1975) Ann. N.Y. 36. Kanazawa, T., Yamada, S., Yamamoto, T., & Acad. Sci. 253, 107-132 Tonomura, Y. (1971) J. Biochem. 70, 95-123 55. Tilney, L.G. & Detmers, P. (1975) J. Cell. Biol. 66, 37. Jorgensen, P.L. (1974) in Methods in Enzymology 508-520 (Colowick, S.P. & Kaplan, N.D., eds.) Vol. 32, pp. 56. Yang, Y. & Perdue, J.F. (1972) J. Biol. Chem. 247, 277-290, Academic Press, New York 4503-4509 38. Shelanski, M.L., Gaskin, F., & Cantor, C.R. (1973) 57. Lazarides, E. & Lindberg, U. (1974) Proc. Natl. Proc. Natl. Acad. Sci. U.S. 70, 765-768 Acad. Sci. U.S. 71, 4742-4746 39. Tietz, A. & Ochoa, S. (1958) Arch. Biochem. Biophys. 58. Hartwig, J.H. & Stossel, T.P. (1975) J. Biol. Chem. 78,477-493 250,5696-5705 40. Boyer, P.D. (1954) J. Amer. Chem. Soc. 76, 4331 59. Tanaka, K., Hayakawa, H., Sakiguchi, M., & Okada, 4337 Y. (1977)Proc. Natl. Acad. Sci. U.S. 74, in press

Vol. 82, No. 1, 1977